Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KC1 with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KC1 concentration is increased above 50 mM KC1. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.     

Eosinophil-derived neurotoxin and human liver ribonuclease. Identity of structure and linkage of neurotoxicity to nuclease activity.

Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.     

Primary structure of an alkaline ribonuclease from bovine liver

A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).     

An improved system for expressing pancreatic ribonuclease in Escherichia coli

An improved method for expressing and purifying bovine pancreatic ribonuclease from a synthetic gene using the lambda promoter controlled by a temperature-sensitive repressor is described. The procedure involves isolation in the presence of a refolding buffer containing oxidized and reduced glutathione, under conditions where RNase can refold, but where proteases presumably do not. Yields are approx. 2 mg purified protein per 1 ferment.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.     

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

Specific RNase isoenzymes in the human central nervous system

After inactivation of RNase inhibitor by parachloromercuribenzoate, total alkaline RNase activity was found to be two fold higher in white matter as in grey matter extracts from human brain tissue. This activity was lower in human purified myelin. Two human cerebrospinal fluid (CSF) RNase isoenzymes of group 3 (a minor one, RNase 3.1, and a major one, RNase 3.2) were found to be present in human grey and white matter extracts and in purified myelin, but absent in human serum, peripheral nerve, liver, and spleen extracts. A RNase isoenzyme similar to central nervous system (CNS) RNase 3.2 was present in human kidney extracts but it differed in its carbohydrate structure. RNase isoenzymes 3.1 and 3.2 were not found in mouse, rat, and bovine brains. Thus, RNases 3.1 and 3.2 seem specific to human CNS. RNases of group 3 are the predominant RNase isoenzymes in CSF and one of the two predominant RNase groups in brain tissue. However, the proportion of RNases of group 3 is different in CSF and in brain extracts: RNases 3.1-3.2 are the major constituents of group 3 RNases in brain tissue, while another RNase isoenzyme of group 3, RNase 3.0, which is more glycosylated than RNases 3.1-3.2, is only a minor part of RNase of group 3 in brain extracts. Conversely, RNases 3.1-3.2 are lower or equivalent to RNase 3.0 in control CSF since the ratio of RNases 3.1-3.2 to RNase 3.0 did not exceed 1.0. This ratio decreased in pathological CSF including multiple sclerosis or infectious CNS diseases that were free of transudation phenomena. In conclusion, CSF RNases 3.1-3.2 seem to originate in brain tissue and could be markers of RNA catabolism from brain cells.     

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

Purification of acid ribonucleases from bovine spleen

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.     

Expression of bovine pancreatic ribonuclease A in Escherichia coli

A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg. RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted. The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A. This work argues that the method suggested by Nagai and Thogersen [Nagai, K. & Thogersen, H. C. (1984) Nature (Lond.) 309, 810-812] for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem. The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis.     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes.

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.     

Transamination of l-cystathionine and related compounds by bovine brain glutamine transaminase

Glutamine transaminase (EC 2.6.1.15) has been purified 113 fold from bovine brain. The product is free of aspariate amino transferase (EC 2.6.1.1.) and other common transaminases. The enzyme shows a wide specificity similar to that reported from the same transaminase purified from bovine kidney and liver as regards both the amino donor and the amino acceptor. Of interest is the transamination and cyclization of l-cystathionine, l-lanthionine, l-cystine and S-aminoethylcysteine. The latter result indicates that the deamination and the cyclization of the sulfur containing diamino acids described for bovine liver and kidney enzyme is feasible also in the brain and suggests the possible endogenous origin of cyclothionine and thiomorpholine dicarboxylate recently detected in bovine brain.     

Purification and properties of bovine kidney ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.     

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : III. The Relationship of the Ribonuclease Activity of Rat Liver Microsomes to their Biological Activity

Attempts have been made to prepare rat liver microsomes and ribosomes free of RNase activity. Washing of microsomes with a large number of reagents, as well as preparation of microsomes by homogenizing the liver in the presence of a variety of reagents chosen to remove or inhibit RNase activity, failed to abolish completely the enzyme activity. However, when rat liver was homogenized in the presence of optimal concentrations of ATP the microsomes subsequently obtained showed no RNase activity. The composition of such microsomes was compared to controls prepared without the use of ATP. Preparation of microsomes with the use of ATP apparently repressed but did not remove the RNase activity for, when such microsomes were treated with 1 per cent deoxycholate to obtain ribosomes, the latter exhibited normal RNase activity. A possible explanation for these results based on several experiments is given. The incorporation of C¹⁴ of L-leucine-C¹⁴ into control and ATP-treated microsomes was measured. Repression of RNase activity by use of ATP or with RNase inhibitor, significantly reduced the incorporation. As a result of these and other experiments it is tentatively concluded that an alkaline RNase is a normal constituent of rat liver ribosomes and plays a role in the biological activity of these particles.     

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.